Novel protein and process for prdoucing the same

ABSTRACT

A protein having amino acid sequence in SEQ ID No.:1 of the Sequence Listing derived from human MP52, and a dimer protein thereof. A homodimer protein described above can be obtained by constructing a plasmid containing DNA coding amino acid sequence in SEQ ID No.:1 of the Sequence Listing with a methionine at the N-terminus, introducing the plasmid into  E. coli  for transformation, solubilizing inclusion bodies obtained by culturing the transformant, purifying the monomer protein from the solubilized solution, refolding the monomer protein into a dimer protein and purifying the same.  
     The homodimer protein described above is useful as a pharmaceutical composition for treating cartilage and bone diseases.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates to a protein having amino acid sequence inSEQ ID No.:1 of the Sequence Listing derived from MP52. The inventionalso relates to a homodimer protein of said protein and a pharmaceuticalcomposition for treating cartilage and bone diseases containing thedimer protein as an active ingredient. The invention also relates to aprocess for preparing the above described protein in a large amount andwith a high purity by culturing E. coli which was transformed with aplasmid containing a DNA sequence capable of expressing the abovedescribed protein. The invention further relates to a method fortreating cartilage and bone diseases, which comprises administering to ahuman a pharmaceutical composition containing, as an active ingredient,an effective amount of the homodimer protein.

[0003] 2. Description of the Prior Art

[0004] Pharmaceutical compositions comprising vitamin D₃, calcitonin,estrogen or their derivatives as well as bisphosphonate derivatives havebeen used in clinical practice for preventing and treating bonediseases. Recently, bone morphogenetic protein (BMP hereinafter), theTGF-β gene superfamily comprising BMP-2 through BMP-9 and relatedproteins, have been reported to have bone morphogenetic activity.

[0005] Furthermore, the bone morphogenetic activity of one of thoseproteins called MP52 has been also reported (WO 93/16099 and WO95/04819). A mature region of MP52 protein is considered to be a proteinconsisting of 120 amino acid residues having the N-terminal alanine, andits amino acid sequence is described in these publications.

[0006] A protein called GDF-5, having an analogous amino acid sequencewith MP52, is also described in Nature, vol. 368, p. 639-643 (1994) andWO 94/15949.

[0007] However, these proteins can not be easily prepared in a purifiedform on an industrial scale.

[0008] Mammalian cell lines such as L-cells have been tried on forproducing MP52 with genetic engineering technology. However, it has beenfound not easy to prepare MP52 in a purified form and in a high yieldwith the expression systems.

DETAILED DESCRIPTION OF THE INVENTION

[0009] The present inventors have tried to prepare MP52 using E. coli ona large scale by genetic engineering technology. Briefly, the inventorshave tried to prepare MP52 using E. coli by adding a codon encodingmethionine to the DNA encoding mature region of MP52 which starts fromalanine. The resultant product was not MP52 only but a mixture of MP52,a protein of 121 amino acid residues having the N-terminal methionineand a protein of 119 amino acid residues having the N-terminal alaninedetached and starting from proline. It was extremely difficult toisolate pure MP52 at least with the mature region from the mixture.

[0010] The inventors have found that a protein in SEQ ID No.:1 of theSequence Listing starting from proline at the N-terminus can beselectively produced in an extremely high yield by constructing aplasmid wherein a codon encoding methionine was connected to the DNAsequence encoding amino acid sequence in SEQ ID No.:1 of the SequenceListing consisting of 119 amino acid residues with elimination of theN-terminal alanine of MP52, and by using the obtained plasmid-introducedE. coli for expression. Moreover, the homodimer of the protein describedin SEQ ID No.:1 in the Sequence Listing was confirmed to have acartilage and bone morphogenetic activity, and thus the invention wascompleted.

[0011] An object of the invention is to provide a protein having aminoacid sequence shown in SEQ ID No.:1 of the Sequence Listing. The proteinconsists of 119 amino acid residues, and corresponds to one in which theN-terminal alanine is eliminated from human MP52 which is regarded as amature region consisting of 120 amino acid residues. The proteinaccording to the invention is soluble in water. Moreover, the protein islow toxic itself because it is derived from human.

[0012] Another object of the invention is to provide a pharmaceuticalcomposition for treating cartilage and/or bone diseases, which comprisesas an active ingredient a homodimer of the protein having the amino acidsequence shown in SEQ ID No.:1 of the Sequence Listing. More in detail,the invention relates to a pharmaceutical composition for preventing andtreating osteoporosis, osteoarthritis such as gonarthritis deformans andmalum coxae deformans, or arthrosteitis, cartilagineous lesion such asarticular meniscus lesion, reconstruction in the defective parts of boneand cartilage caused by injury and oncoectomy, defect of bone andcartilage, bone fracture, congenital cartilage and bone diseases such aschondrodysplasia, chondrohypoplasia, achondrogenesis, palatoschisis andosteodysplasia, and furthermore radicular and arvecular defects, sincethe homodimer protein according to the invention has a cartilage andbone morphogenetic activity. Furthermore, the homodimer protein can beapplied to a treatment of bone grafting in cosmetic surgery. Thesetreatments also include those in the area of veterinary surgery.

[0013] A further object of the invent on is to provide a process forpreparing a protein consisting of 119 amino acid residues derived fromhuman MP52 shown in SEQ ID No.:1 of the Sequence Listing using E. coli.

[0014] In particular, the invention relates to the construction of aplasmid containing a DNA sequence encoding the amino acid sequenceconsisting of 119 amino acid residues shown in SEQ ID No.:1 of theSequence Listing with an additional methionine at the N-terminus. Onlythe mature region of human MP52 cDNA was amplified by polymerase chainreaction (PCR method) by using, as a template DNA, a plasmid vectorcontaining cDNA described in WO 93/16099. The PCR method referred toherein generally means to multiply a very small amount of fragments ofDNA or RNA by the method described in U.S. Pat. NO. 4,683,195.

[0015] It is necessary for preparing the protein of the invention toconstruct appropriate expression vectors containing DNA encoding theprotein, which are then introduced into desirable E. coli host strainsby genetic engineering technology. The following two improved processeswere applied for a large scale production of the protein;

[0016] 1) A process or increasing the productivity of target proteins byincreasing the translation efficiency as reported by M. Nobuhara et al.{Agric. Biol. Chem., 52 (6), 1331-1338, 1988}, viz. the method ofincreasing the AT content around the ATG initiation codon, and

[0017] 2) A process for increasing an average copy number of plasmidsper cell, viz. the method of replacing ori region for the replicationorigin of pBR vector by that of pUC vector. Further, the expressionvector (pKOT245) of the invention was constructed by direct ligation ofthe promoter region with the DNA sequence encoding amino acid sequencein SEQ ID No.:1 of the Sequence Listing with an additional methionine inits N-terminus. The E. coli containing said vector was deposited(Accession No. BIKOKEN-KI P-14895) at National Institute of Bioscienceand Human-Technology, Agency of Industrial Science and Technology whichis located at 1-3, Higashi 1-chome, Yatake-cho, Tsukuba-shi,Ibaraki-ken, 305 Japan on Apr. 14, 1995 and transferred to a deposit(Accession No. BIKOKEN-KI BP-5499) on Apr. 10, 1996 according toBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms.

[0018] This invention relates to a process for preparing monomerproteins comprising the steps of:

[0019] constructing a plasmid containing DNA encoding amino acidsequence in SEQ ID No.:1 of the Sequence Listing with a methionine atits N-terminus,

[0020] introducing the plasmid into E. coli for transformation,

[0021] cultivating the E. coli to obtain inclusion bodies,

[0022] solubilizing and purifying said inclusion bodies to obtainmonomer proteins, and

[0023] to a process for preparing homodimer proteins of the protein inSEQ ID No.:1 of the Sequence Listing by refolding and purifying themonomer proteins obtained in the above. Briefly, the proteins of theinvention were prepared by solubilizing the E. coli inclusion bodiesfollowed by loading on SP-Sepharose FF column and Sephacryl S-200 columnto obtain purified sulfonated MP52 monomers, which were subjected torefolding and isoelectric precipitation, then to RESOURCE RPC column ofreverse-phase HPLC to obtain the purified dimer fractions of theproteins. The physicochemical properties of the proteins were analyzedon the basis of N-terminal amino acid sequence and amino acidcomposition and by electrophoresis.

[0024] This invention further relates to a process for culturing E. coliwhich were introduced with the expression vectors of the invention underthe conditions of culture medium at 28-34° C., pH 6-8 and a dissolvedoxygen concentration of 20-50%.

[0025] This invention further relates to a method for treating cartilageand bone diseases, which comprises administering to a human apharmaceutical composition containing, as an active ingredient, aneffective amount of the homodimer protein.

[0026] Biological activities of the homodimer protein were determined byanalysis of soft X-ray radiographs, analysis of tissue-staining andanalysis of time-course of ectopic cartilage/bone formation.Furthermore, from the results of the effect on the intramembranousossification, the effect on the regeneration of articular cartilage andthe effect on the healing of bone fracture and defects, the homodimerprotein of the present invention is proved to be beneficial to thetherapies of cartilage and/or bone regeneration.

[0027] The homodimer protein of the invention can be administered insystemic by intravenous, intramuscular or intra-peritoneal injection. Incase of intravenous administration, an intravenous drip infusion canalso be used, in addition to conventional intravenous injections.

[0028] Injectable preparations can be formulated, for example, in theform of injectable powders. In that case, the powders can be prepared byadding one or more of suitable water-soluble excipients such asmannitol, sucrose, lactose, maltose, glucose, fructose and the like, toan active ingredient, dissolving the mixture in water, dividing it intovials or ampoules followed by lyophilizing and hermetically sealing.

[0029] In the case of local administration, the homodimer protein can becoated on the surface of cartilage, bone or tooth to be treated withcollagen paste, fibrin glue or other adhering materials. In the case ofbone grafting, both natural bone and conventional artificial bone can beused. Artificial bone means the bone made of metal, ceramics, glass, andother natural or artificial inorganic substance. Hydroxyapatite is citedas preferable artificial substance. For example, artificial bone can beconstructed by steel as dense material in the inner part andhydroxyapatite as porous material in outer part. Moreover, it isbeneficial to apply the homodimer protein to the part from whichcancerous bone tissue is removed in order to accelerate thereconstruction of bone. It can also be applied to the cartilagegrafting.

[0030] The dose may be varied depending upon various factors influencingthe activity of the protein such as weight of bone and cartilage to bereconstructed, injured site of bone and cartilage and the symptoms, ageand sex of patients, severity of infection, administration intervals andother clinical factors. The dose can also be varied depending upon typesof carriers to be used for restructuring with the dimer protein. Ingeneral, the dose is in the range of about 10-10⁶ ng of the homodimerprotein per wet weight of desired bone and cartilage when administeredas a composition containing a carrier. In the case of local and systemicadministration by injection, it is preferable to administer 0.1-10⁴ μgin a frequency of from once a week to once a day.

[0031] A synergetic effect can be expected by administering thehomodimer protein simultaneously with known growth factors, for example,insulin-like growth factor-I for regeneration of bone and cartilage.

[0032] There has never been reported of a process for preparing theprotein of the invention on an industrial scale and in a purified formas described above, and the homodimer protein is useful as a medicalcomposition for treating cartilage and bone diseases since it has acartilage and bone morphogenetic activity. Further, the process ofpreparing the protein of the present invention can be applicable for thepreparation of other proteins of the above-described TGF-β superfamilymembers, all of which were only successful so far to prepare by usingmammalian cell lines.

[0033] This invention is further Illustrated by the following examples.However, it should not be construed that the invention is limited tothese specific examples.

EXAMPLE Example 1 Construction of Expression Vector

[0034] (1) Isolation of a mature region of MP52

[0035] A mature region of human MP52 cDNA was PCR-amplified using theplasmid vector (pSK52s) containing cDNA described in WO 93/16099 as atemplate DNA.

[0036] In accordance with the process for increasing a productivity ofthe target proteins reported by M. Nobuhara, et al. {Agric. Biol. Chem.,52 (6), 1331-1338, 1988}, a part of DNA of the mature region of MP52gene was substituted to increase the AT content around the ATGinitiation codon.

[0037] The mutagenesis was introduced by PCR method using the designedupstream PCR primer encompassing the mutation of SEQ ID No.:2 of theSequence Listing. For the DNA sequence of the PCR primers were used theDNA in the SEQ ID No.:2 as an upstream primer, and the DNA in SEQ IDNo.:3 of the Sequence Listing as a downstream primer.

[0038] The PCR was performed by adding the template DNA (10 ng), 50pmols each of the PCR primers in an order direction and in a reversedirection, dNTP (0.2 mmol) and MgCl₂ (1.5 mmol) in the same test tube,together with Taq DNA polymerase (5 U).

[0039] Thirty cycles of PCR were performed; the conditions of each cyclewere 94° C. for a minute for denaturation, 55° C. for a minute forprimer annealing, and 72° C. for 2 minutes for primer extension.

[0040] The products obtained from the PCR was isolated byelectrophoresis in 1.5% low melting point agarose (purchased from FMC),and the fragments of about 360 bp were isolated (Fragment 1).

[0041] (2) Construction of E. coli expression vector for the protein ofthe invention

[0042] In order to increase a copy number of the plasmid per bacteria,the ori region for replication origin was changed from that of pBR topUC vector. The E. coli expression vector pKK223-3 available in themarket (purchased from Pharmacia Biotech) was used to isolate tacpromoter region by digestion with restriction endonucleases SspI andEcoRI, and also to isolate rrnBt₁t₂ terminator region by using SalI andSspI. A DNA fragment of tac promoter region which had been treated withMung Bean Nuclease (Takara Shuzo Co., Ltd.) was ligated by T4 DNA ligasewith Fragment 1 which was obtained above. The resultant DNA fragment wasdigested by SalI and re-ligated with the rrnBt₁t₂ region. The DNAfragment was ligated into the SmaI site of pUC18 vector to construct theexpression vector {pKOT245 (Accession No. BIKOKEN-KI P-14895)} (FIG. 1)for the production of the protein. The length of pKOT245 DNA is 3.7 kb.The nucleotide sequence of the expression vector constructed for theprotein was analyzed by Pharmacia ALF DNA sequencer.

[0043] (3) Transformation

[0044] Transformation was performed according to the rubidium chloridetransformation method by Kushner et al. (Genetic Engineering, p. 17,Elsevier, 1978). Briefly, pKOT245 was used to transform the host strainE. coli W3110M according to the method described above to produce E.coli transformants for the production of the protein.

Example 2 Cultivation

[0045] (1) Cultivation

[0046] The E. coli expressing the protein of the invention wasprecultured in the modified SOC medium (Bacto tryptone 20 g/l, Bactoyeast extract 5 g/l, NaCl 0.5 g/l, MgCl₂.6H₂O 2.03 g/l, Glucose 3.6g/l). 100 ml of the bacteria suspension was used to inoculate 5 l of theproduction medium (Bacto tryptone 5 g/l, Citric acid 4.3 g/l, K₂HPO₄4.675 g/l, KH₂PO₄ 1.275 g/l, NaCl 0.865 g/l, FeSO₄.7H₂O 100 mg/l,CuSO₄.5H₂O 1 mg/l, MnSO₄.nH₂O 0.5 mg/l, CaCl₂.2H₂O 2 mg/l, Na₂B₄O₇.10H₂O0.225 mg/l, (NH₄)₆Mo₇O₂₄.4H₂O 0.1 mg/l, ZnSO₄.7H₂O 2.25 mg/l, CoCl₂.6H₂O6 mg/l, MgSO₄.7H₂O 2.2 g/l, Thiamine HCl 5.0 mg/l, Glucose 3 g/l), whichwas cultured in a 10-liter fermentor with aeration-agitation, and thenupon reaching the early stage of logarithmic growth phase (OD₅₅₀=5.0),isopropyl-3-D-thio-galactopyranoside at a final concentration of 1 mMwas added and the cultivation was continued until reaching OD₅₅₀=150.During the cultivation, temperature was kept at 32° C., and pH value of7.15 by adding ammonia. In order to prevent lowering of a dissolvedoxygen concentration, an agitation was sped up to keep the dissolvedoxygen concentration at 50% of air saturation. The cultivation wasproceeded by adding 50% glucose solution at a level of 0.2% to obtain ahigh cell density, with an indication of abrupt increase of thedissolved oxygen concentration.

[0047] (2) Preparation of E. coli inclusion bodies

[0048] The culture broth obtained by the method described above wascentrifuged to harvest the cells, which were then suspended in 25 mMTris-HCl buffer containing 10 mM ethylene diamine tetraacetic acid (pH7.3). The cells were disrupted by passing through a homogenizer (made byAPV Gaulin Inc.) and centrifuged again to harvest the precipitatecontaining the inclusion bodies.

Example 3 Purification

[0049] (1) Solubilization of E. coli inclusion bodies

[0050] After washing with 1% Triton X-100 three times, the E. coliinclusion bodies were centrifuged at 3,000×g for 30 minutes at 4° C.,and then the resultant precipitate was solubilized by sonication in 20mM Tris-HCl buffer, pH 8.3, 8 M urea, 10 mM DTT, and 1 mM EDTA.

[0051] (2) Preparation of monomers

[0052] The solubilized solution was centrifuged at 20,000×g for 30minutes at 4° C. and the resultant supernatant was collected. Theobtained supernatant was subjected to SP-Sepharose FF (Pharmacia AB)equilibrated with 20 mM Tris-HCl buffer pH 8.3, 6 M urea, and 1 mM EDTA,and then, after washing with the same solution, it was eluted with thesame solution containing 0.5 M NaCl. The protein in the eluate weresulfonated by adding Na₂SO₃ and Na₂S₄O₆ to read the final concentrationrespectively at 111 mM and 13 mM and by incubating at 4° C. for 15hours. The sulfonated solution was gel-filtrated on Sephacryl S-200 HR(Pharmacia AB) equilibrated with 20 mM Tris-HCl buffer, pH 8.3, 6 Murea, 0.2 M NaCl, and 1 mM EDTA to obtain purified sulfonated monomersof the protein of the invention.

[0053] (3) Refolding

[0054] The solution of the sulfonated monomers was added into a 9 timesvolume of 50 mM Na-Glycine buffer pH 9.8, 0.2 M NaCl, 16 mM CHAPS, 5 mMEDTA, 2 mM GSH (reduction type glutathione), and 1 mM GSSG (oxydationtype glutathione) with stirring, and then incubated or 24 hours at 4° C.to oxidize and refold the protein of the invention.

[0055] (4) Preparation of homodimers

[0056] The refolding solution was diluted with the same volume ofpurified water and then by adding 6 N NaCl adjusted pH value toapproximately 7.4 and placed to isoelectric precipitation. Theprecipitates collected by centrifugation at 3,000×g for 20 minutes weresolubilized in a solution with 30% acetonitrile containing 0.1% TFA. Thesolution was diluted with the same volume of purified water and loadedon RESOURCE RPC column (Pharmacia AB) of a reverse-phase HPLCpreequilibrated with 25% acetonitrile containing 0.05% TFA, and theneluted with a linear gradient of 25-45% acetonitrile containing 0.05%TFA. The eluate was monitored at 280 nm absorbance. The purifiedhomodimer protein fractions were collected and lyophilized by SpeedVacConcentrator (Servant Co.)

[0057] (5) Determination of physicochemical properties of the purifiedprotein of the invention

[0058] a) Analysis of N-terminal amino acid sequence

[0059] Analysis of the N-terminal amino acid sequence for the purifiedproteins was performed using an amino acid sequencer Model 476A (AppliedBiosystems Inc.) to confirm the amino acid sequence from the N-terminalto the 30th amino acid as shown in SEQ ID No.:1 of the Sequence Listing.

[0060] b) Analysis or amino acid composition

[0061] The analysis of amino acid composition of the purified proteinsobtained above was performed by an amino acid sequencer (PICO TAGSystems, Waters). The result was shown in Table 1. The number describedin Table 1 indicates the number of amino acid residue per a monomerprotein. TABLE 1 Amino acid Practical number Expected number Asx 11.5 12Glx 10.9 11 Ser 8.4 9 Gly 4.3 4 His 4.0 4 Arg 7.7 7 Thr 5.4 6 Ala 7.3 7Pro 10.2 10 Tyr 2.9 3 Val 5.7 7 Met 5.1 4 ½ Cys 2.6 7 Ile 4.9 6 Leu 10.010 Phe 4.0 4 Lys 5.9 6 Typ — 2 length of 119 the sequence

[0062] c) Analysis by electrophoresis

[0063] Molecular weight or the purified proteins obtained above wasconfirmed to be about 28 KDa on SDS-PAGE under non-reducing condition.

[0064] From the results shown in the above a), b) and c), it is foundthat the protein of the invention comprises 119 amino acid residuesstarting from the N-terminal Pro singly.

Example 4 Determination of Biological Activities

[0065] (1) Activity in ectopic bone formation in mice

[0066] About 500 μg of the homodimer protein obtained in Example 3 wasdissolved and diluted in 50 μl of 10 mM hydrochloric aced, and 1 μg/10μl, 10 μg/10 μl, and 100 μg/10 μl concentrations of the solution wereprepared. Ten μl of each solution was mixed with 150 μl porcine tendontype-I collagen solution (Koken, 0.5%, pH 3, I-AC), neutralized,lyophilized, and the resultant mixture was implanted into pocketscreated in the thigh muscles of 8-week-old male ICR mice. At day 21 fromimplantation, the animals were sacrificed and the thighs were excised.After peeling skins off, the incidence of calcified tissues wasevaluated by soft X-ray radiography. As shown in Table 2, theimplantation of 1 μg/site or more of the dimer protein induced calcifiedtissue in part of the group of the mice, and 10 and more doses inducedcalcified tissue in all mice used. TABLE 2 Dose of the homodimer proteinIncidence of calcified tissue Control (Type-I collagen alone) 0/4  1μg/site 3/4  10 μg/site 4/4 100 μg/site 4/4

[0067]FIG. 2 shows typical examples of soft X-ray radiographs ofcalcified tissue induced by different doses of MP52 protein. FIGS. 2A,2B and 2C show examples of soft X-ray radiographs of 1 μg the homodimerprotein/site-, 10 μg/site- and 100 μg/site-implanted mice thighs,respectively. These radiographs indicate that the homodimer proteininduced calcified tissue in the mouse thigh and increased it in adose-dependent manner. In order to verify if the formed calcifiedtissues were cartilage or bone, the sections of the fixed mouse thighsinto which 10 μg/site the homodimer protein was implanted were stainedwith von Kossa, Alcian blue or Hematoxylin-eosin.

[0068]FIG. 3 shows light-microscopic photographs of the sections stainedwith the respective staining methods. In FIG. 3A (von Kossa staining),areas indicated by ct and cc show calcified tissue and calcifiedchondrocytes, respectively. In FIG. 3B (Alcian blue staining), an areaindicated by rc shows remaining cartilage tissue. In FIG. 3C(Hematoxylin-eosin staining), elements indicated by ad, bm, lb, ob andwb are an adipocyte, bone marrow cells, lamellae bone, osteoblasts, andwoven bone, respectively. Thus, it is evident that the implantation ofthe homodimer protein with Type-I collagen into mouse thighs inducescalcified chondrocytes, osteoblasts, and bone marrow cells at the sites.

[0069] Thus, the homodimer protein was demonstrated to possess activityin ectopic cartilage and bone formation.

[0070] (2) Analysis of time-course of ectopic bone formation in mice

[0071] The dimer protein (3 μg) obtained in Example 3 was mixed withType-I collagen solution and neutralized as described in Example 4 (1),and the lyophilized materials were implanted into the male ICR mousethighs. At days 3, 7, 10, 14, 21, and 28 from implantation, the thighswere excised and fixed in 10% formalin, and then, sections were stainedwith Hematoxylin-eosin or von Kossa. FIG. 4 shows the light-microscopicphotographs of the sections stained.

[0072] At day 3 (FIG. 4A, Hematoxylin-eosin staining), undifferentiatedmesenchymal cells (mc) including morphologically fibrous connectivecells appeared in the space between collagen fibers (co) implanted andmuscle cells (m). At between days 7 and 10 FIGS. 4B and 4C,respectively, Hematoxylin-eosin staining), the space was filled withundifferentiated mesenchymal cells (mc) and these cells werehypertrophied and differentiated into precartilagenous tissue. At day 14(FIG. 4D, Hematoxylin-eosin staining and FIG. 4E, von Kossa staining),calcified cartilage tissue (cc) and bone tissue (b) were observed. Atday 21 (FIG. 4D, Hematoxylin-eosin staining and FIG. 4E, von Kossastaining), calcified cartilage tissue was not observed at all, and thetissue observed at day 14 appeared to be replaced into bone (b) withbone marrow (bm). At day 28 (FIG. 4H, Hematoxylin-eosin staining), therewere a large mount of bone marrow cells and formed bone appeared to beunder a resorptive process.

[0073] Thus, it is evident that the homodimer protein inducesendochondral ossification through cartilage formation at ectopic sites,as reported by using other BMPs.

[0074] (3) Effect on the intramembranous ossification

[0075] The homodimer protein obtained in Example 3 was dissolved inphosphate-buffered saline (pH 3.4 ) containing 0.01% human serumalbumin, and 0.01 μg/20 μl-, 0.1 μg/20 μl-, and 1 μg/20μl-concentrations of solutions were prepared. Twenty μl-portion of eachsolution was injected 12 times once a day onto the periosteum ofneonatal rat parietal bone by using a microsyringe from day 1 afterbirth. The same volume of the vehicle was injected onto the counter-sideof parietal bone of each rat. The same volume of the vehicle was alsoinjected onto both sides of parietal bones of control rats. At day 1from the final injection, the rats were sacrificed and the both sides ofparietal bones were excised and fixed, and then, the decalcifiedsections stained with Hematoxylin-eosin were prepared to measure thethickness of the parietal bones at the injected sites on microscopicphotographs. The ratio of the homodimer protein-injectedsite/vehicle-injected site in the parietal bone thickness of each ratwas calculated. As shown in Table 3, the homodimer protein increasedparietal bone thickness in a dose-dependent manner. A typical example ofmicroscopic photographs of the section at a homodimer protein 0.1μg/site-injected site is shown in FIG. 5B in comparison with that of thecounter-side of vehicle-injected site (FIG. 5A). The injection of thehomodimer protein induced the activation and proliferation of periostealcells (p), and activated osteoblasts (ob) were observed in and on theparietal bone (b). These results indicate that the homodimer proteinstimulated intramembranous ossification when locally injected, and thatthe homodimer protein is beneficial to the therapies of osteoporosis,bone fracture, and alveolar ridge and periodontal defects. TABLE 3 Doseof Parietal bone thickness (μn) homodimer protein vehicle-injectedMP52-injected Ratio protein (μg/site/day) site (A) site (B) (B/A) 0(vehicle) 128 ± 7 141 ± 20 1.10 ± 0.16 0.01 134 ± 9 167 ± 30 1.27 ± 0.330.1  119 ± 19 190 ± 29  1.60 ± 0.10* 1 132 ± 9 225 ± 25  1.70 ± 0.14**

[0076] (4) Effect on the regeneration of articular cartilage

[0077] Six 12-week-old male New Zealand White rabbits were used for thisstudy. Right knee skin and articular capsule were cut and a 5×5 mm fullthickness osteochondral defect was created in the patellar groove usinga dental burr so as not to damage surrounding tendons. The defects werefilled with either lyophilized Type-I collagen sponge or withlyophilized Type-I collagen sponge containing 10 μg homodimer protein,prepared as described in Example 4 (1), and then, the cut articularcapsule and skin were sutured. Three weeks post-operatively, the rabbitswere sacrificed and the femoral heads were excised and fixed in 10%formalin, and then, decalcified sections were stained with Alcian blue.Typical examples of microscopic photographs of the sections were shownin FIG. 6. The dimer protein treated defects (FIGS. 6C and 6D)demonstrated the regeneration of chondrocytes (ch) with extracellularmatrices which were stained intensively with Alcian blue, as compared tothe Type-I collagen sponge implanted control defects (FIGS. 6A and 6B)which were filled with fibrous tissue (f). The cartilage tissue inducedby the dimer protein showed zonal structure including restingchondrocytes, growing chondrocytes and hypertrophied chondrocytes, likethat of normal articular cartilage. The chondroinduction by the MP52protein were observed in the defects of all rabbits used (n=3). Theseresults indicate that the dimer protein is effective to the repair ofdamaged cartilage tissue in patients such as osteoarthritis.

[0078] (5) Effect on the healing of bone fracture and defects

[0079] Thirty male Sprague-Dawley rats (about 15-week-old) were used forthis study. Using a lateral approach to the femur, all muscle andperiosteal tissue were stripped from the diaphysis. A 5 mm-segmentalbone defect was created in the middle region of the right femur shaftwith use of dental burr, and then, a special-made polyethylene plate wasfixed with stainless screws along the cortex of the femur. Type-Icollagen sponges containing 0, 1, 10, and 100 μg of the homodimerprotein were prepared as described in Example 4 (1), and implanted intothe segmental bone defects and then, the wound was sutured. Just afteroperation and 12 weeks post-operatively, the defects were evaluated bysoft X-ray radiography. As shown in FIG. 7, 10 and 100 μg/site of thehomodimer protein stimulated callus (cs) formation in the defects andformed bony unions, but the effect at 1 μg/site was not clear ascompared to the control collagen implanted defect in which only marginalendosteal bone formation was observed. Twelve weeks post-operatively,rats were sacrificed, and the femur with a defect was excised and bonemineral content (accumulated one of mid-three scannings in the defect)was measured by dual energy X-ray absorptiometry (Aloka, Model DCS-600)in a mode with 1 mm scanning width after removing the polyethyleneplate. Both ends of the femur with the resin were fixed, then, maximumtorsional strength to break the union of specimens were measured by bonestrainer system (Malto, model MZ-500D) in a routing speed with 180°/min(Table 4). It shows that the homodimer protein increases both bonemineral content and bone strength at the rat femur defect in which theprotein is implanted, and indicates the efficacy of the present proteinfor fracture healing and bone reconstruction of the defect. TABLE 4 Doseof Bone Mineral Maximum Torsional homodimer pro- Content in rat Strengthtein (μg/site) femur defect (mg) (Kgf · cm) Number collagen alone 120.2± 24.5 2.92 ± 0.09 6 1 176.9 ± 36.4 6.24 ± 1.00 8 10 277.4 ± 63.9 9.35 ±3.14 8 100  374.8 ± 67.1* 40.34 ± 7.64* 8

[0080] From the results in Example 4, the homodimer protein of theinvention was found to have a cartilage and bone morphogenetic activity.

[0081] The protein composed of a homodimer of the protein having anamino acid sequence in SEQ ID No.:1 of the Sequence Listing has acartilage and bone morphogenetic activity and is useful as apharmaceutical composition for treating cartilage and bone diseases.Furthermore, the protein of the invention can be prepared on anindustrial scale and in a pure form by a gene engineering processculturing E. coli transformed with a higher copy number expressionvector for said protein.

BRIEF EXPLANATION OF DRAWINGS

[0082]FIG. 1 shows a plasmid map of the expression vector (pKOT245) forthe protein of the invention obtained in Example 1 (2).

[0083]FIG. 2 shows a soft X-ray radiograph of the calcified tissueinduced in mouse thigh in Example 4 (1).

[0084]FIG. 3 shows a light-microscopic photograph of the calcifiedtissue stained in mouse thigh in Example 4 (1).

[0085]FIG. 4 shows a light-microscopic photograph of the time-coursedcalcified tissue stained in mouse thigh in Example 4 (2).

[0086]FIG. 5 shows a light-microscopic photograph of rat parietal bonestained in Example 4 (3).

[0087]FIG. 6 shows a light-microscopic photograph of articular cartilagedefects stained in the rabbit femoral head in Example 4 (4).

[0088]FIG. 7 shows a soft X-ray radiograph of the bone formation in thebone defects of the rat femurs in Example 4 (5).

1 4 119 AMINO ACIDS AMINO ACID LINEAR PEPTIDE HOMOSAPIENS FETUS MP52 383TO 501 1 Pro Leu Ala Thr Arg Gln Gly Lys Arg Pro Ser Lys 1 5 10 Asn LeuLys Ala Arg Cys Ser Arg Lys Ala Leu His 15 20 Val Asn Phe Lys Asp MetGly Trp Asp Asp Trp Ile 25 30 35 Ile Ala Pro Leu Glu Tyr Glu Ala Phe HisCys Glu 40 45 Gly Leu Cys Glu Phe Pro Leu Arg Ser His Leu Glu 50 55 60Pro Thr Asn His Ala Val Ile Gln Thr Leu Met Asn 65 70 Ser Met Asp ProGlu Ser Thr Pro Pro Thr Cys Cys 75 80 Val Pro Thr Arg Leu Ser Pro IleSer Ile Leu Phe 85 90 95 Ile Asp Ser Ala Asn Asn Val Val Tyr Lys Gln Tyr100 105 Glu Asp Met Val Val Glu Ser Cys Gly Cys Arg 110 115 27 BASEPAIRS NUCLEIC ACID SINGLE LINEAR 2 ATAATGCCAC TAGCAACTCG TCAGGGC 27 26BASE PAIRS NUCLEIC ACID SINGLE LINEAR 3 CGTCGACTAC CTGCAGCCAC ACGACT 26357 BASE PAIRS NUCLEIC ACID DOUBLE UNKNOWN 4 CCA CTG GCC ACT CGC CAG GGCAAG CGA CCC AGC AAG 36 Pro Leu Ala Thr Arg Gln Gly Lys Arg Pro Ser Lys 15 10 AAC CTT AAG GCT CGC TGC AGT CGG AAG GCA CTG CAT 72 Asn Leu Lys AlaArg Cys Ser Arg Lys Ala Leu His 15 20 GTC AAC TTC AAG GAC ATG GGC TGGGAC GAC TGG ATC 108 Val Asn Phe Lys Asp Met Gly Trp Asp Asp Trp Ile 2530 35 ATC GCA CCC CTT GAG TAC GAG GCT TTC CAC TGC GAG 144 Ile Ala ProLeu Glu Tyr Glu Ala Phe His Cys Glu 40 45 GGG CTG TGC GAG TTC CCA TTGCGC TCC CAC CTG GAG 180 Gly Leu Cys Glu Phe Pro Leu Arg Ser His Leu Glu50 55 60 CCC ACG AAT CAT GCA GTC ATC CAG ACC CTG ATG AAC 216 Pro Thr AsnHis Ala Val Ile Gln Thr Leu Met Asn 65 70 TCC ATG GAC CCC GAG TCC ACACCA CCC ACC TGC TGT 252 Ser Met Asp Pro Glu Ser Thr Pro Pro Thr Cys Cys75 80 GTG CCC ACG CGA CTG AGT CCC ATC AGC ATC CTC TTC 288 Val Pro ThrArg Leu Ser Pro Ile Ser Ile Leu Phe 85 90 95 ATT GAC TCT GCC AAC AAC GTGGTG TAT AAG CAG TAT 324 Ile Asp Ser Ala Asn Asn Val Val Tyr Lys Gln Tyr100 105 GAG GAC ATG GTC GTG GAG TCG TGT GGC TGC AGG 357 Glu Asp Met ValVal Glu Ser Cys Gly Cys Arg 110 115

What is claimed is:
 1. A protein having amino acid sequence in SEQ IDNo.:1 of the Sequence Listing.
 2. A homodimer protein according toclaim
 1. 3. A pharmaceutical composition for treating cartilage and bonediseases comprising a therapeutically effective amount of the proteinaccording to claim 2 and a pharmaceutical carrier.
 4. The pharmaceuticalcomposition according to claim 3 wherein cartilage and bone diseases areosteoporosis.
 5. The pharmaceutical composition according to claim 3wherein cartilage and bone diseases are osteoarthritis or arthrosteitis.6. The pharmaceutical composition according to claim 3 wherein cartilageand bone diseases are bone fracture and bone defect.
 7. Thepharmaceutical composition according to claim 3 wherein cartilage andbone diseases are radicular and arvecular defects.
 8. A process forpreparing the protein of claim 1 which comprises culturing E. colitransformed with a plasmid containing DNA sequence which is capable ofexpressing said protein.
 9. The process according to claim 8 wherein theplasmid contains a DNA coding amino acid sequence in SEQ ID No.:1 of theSequence Listing with a methionine at the N-terminus.
 10. A process forpreparing the dimer protein according to claim 2 which comprises thesteps of; constructing a plasmid containing DNA coding amino acidsequence in SEQ ID No.:1 of the Sequence Listing with a methionine atthe N-terminus, introducing the plasmid into E. coli for transformation,solubilizing inclusion bodies obtained by culturing said E. coli,purifying the monomer protein from the solubilized solution, refoldingthe monomer protein into a dimer protein and purifying the same.
 11. Amethod for treating cartilage and bone diseases, which comprisesadministering to a human a pharmaceutical composition containing, as anactive ingredient, an effective amount of the homodimer protein asclaimed in claim
 2. 12. The method for treating cartilage and bonediseases as claimed in claim 11, wherein said disease is osteoporosis.13. The method for treating cartilage and bone diseases as claimed inclaim 11, wherein said disease is osteoarthritis or arthrosteitis. 14.The method for treating cartilage and bone diseases as claimed in claim11, wherein said disease is bone fracture and bone defect.
 15. Themethod for treating cartilage and bone diseases as claimed in claim 11,wherein said disease is radicular and arvecular defects.